A general problem in molecular genetics and synthetic biology is the construction of concatenated sets of DNA fragments. The DNA fragments can represent genome segments, individual genes, domains within genes, etc. In some cases it is useful to shuffle the fragments (possibly from a large pool of input fragments) to generate random sets. In other cases it is useful to program the order in which DNA fragments are concatenated. In general, it is useful to have the ability to both shuffle and program the fragments and their arrangement as projects move from hypothesis generation to hypothesis testing phases. The DNA fragments assembled in such sets can be used in gain-of-function experiments to construct alternative metabolic pathways (Shao et al. 2008), multi-protein complexes, virulence systems that involve concerted attack on host defenses, or in loss-of-function experiments involving RNA interference where multiple genes redundantly contribute to a phenotype (Zhu et al., “A Versatile Approach to Multiple Gene RNA Interference Using MicroRNA-Based Short Hairpin RNAs,” BMC Mol. Biol. 8:98 (2007)).
A general method for concatenating DNA fragments that lack sequence homology is to use various methods (primarily PCR) to attach short adapters to the ends of the DNA fragments to be concatenated (and typically inserted into a vector in the same process). These flanking “adapters” can recombine in yeast (Raymond et al., “General Method for Plasmid Construction Using Homologous Recombination,” Biotechniques 26:134-8, 140-1 (1999)) or in bacterial strains expressing phage recombinases (Bieniossek et al. “Automated Unrestricted Multigene Recombineering for Multiprotein Complex Production,” Nat. Methods 6:447-50 (2009)) that support recombination of such short (ca. 30 bp) adapters. This technology of adapter-driven recombination of DNA fragments in yeast is robust, and was used for example, to form a complete synthetic Mycoplasma genitalium genome (Gibson et al., “One-Step Assembly in Yeast of 25 Overlapping DNA Fragments to Form a Complete Synthetic Mycoplasma Genitalium Genome,” Proc. Nat'l. Acad. Sci. USA 105:20404-9 (2008)). A disadvantage of this method is that DNA fragments can only assemble in the manner directed by attached adapters (i.e., programmed assembly) and alternative assemblies require the generation of DNA fragments with different adapter arrangements. In other words, each DNA fragment must be specialized to achieve a specific assembly. This approach becomes very laborious when trying to assemble several DNA fragments into several different genetic constructs.
In an extension of this approach, target DNA and vector fragments were electroporated into yeast along with 80-bp “linker” oligonucleotides carrying homology with the target fragment and the vector. Without further experimental manipulation, yeast recombined these into the desired construct (Raymond et al., “Linker-Mediated Recombinational Subcloning of Large DNA Fragments Using Yeast,” Genome Res. 12:190-7 (2002)). Although this approach can be used to generate concatenated sets of DNA fragments in a designed arrangement, it is unlikely to work for shuffling fragments. Further, the DNA fragments can only assemble in the manner directed by the co-transfected linker oligonucleotides and alternative assemblies requires the generation and use of different linker oligonucleotides.
A particularly powerful, recent application of this general approach is the “Golden Gate Shuffling” method as described by Engler et al, “Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes” PLoS One 4:e5553 (2009), which involves constructing DNA fragments terminated with a unique sequence of four nucleotides followed by a BsaI cleavage site. Cleavage with BsaI exposes the four nucleotides as a single-stranded overhang that can hybridize with the overhang of another DNA fragment, as designed. A general limitation with golden gate shuffling (Engler et al., “Golden Gate Shuffling: a One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes,” PLoS One 4:e5553 (2009)), or in vitro sequence and ligation-independent cloning (SLIC) (Li and Elledge, “Harnessing Homologous Recombination in vitro to Generate Recombinant DNA Via SLIC,” Nat. Methods 4:251-6 (2007)), or yeast-based recombination systems such as “DNAassembler” (Shao et al., “DNA Assembler, an in vivo Genetic Method for Rapid Construction of Biochemical Pathways,” Nucl. Acids Res. (in press) (2008)), is that the generation of assemblies that are shuffled involves alternative arrangements of concatenated DNA fragments, which requires the generation and maintenance of multiple variants (differing in adapters) of each DNA fragment in the set.
The present invention is directed to overcoming these and other deficiencies in the art.